Non-pandemic Strains Research Articles

The Identification Distinct Antiviral Factors Regulated Influenza Pandemic H1N1 Infection.

Influenza pandemic with H1N1 (H1N1pdms) causes severe lung damage and "cytokine storm," leading to higher mortality and global health emergencies in humans and animals. Explaining host antiviral molecular mechanisms in response to H1N1pdms is important for the development of novel therapies. In this study, we organised and analysed multimicroarray data for mouse lungs infected with different H1N1pdm and nonpandemic H1N1 strains. We found that H1N1pdms infection resulted in a large proportion of differentially expressed genes (DEGs) in the infected lungs compared with normal lungs, and the number of DEGs increased markedly with the time of infection. In addition, we found that different H1N1pdm strains induced similarly innate immune responses and the identified DEGs during H1N1pdms infection were functionally concentrated in defence response to virus, cytokine-mediated signalling pathway, regulation of innate immune response, and response to interferon. Moreover, comparing with nonpandemic H1N1, we identified ten distinct DEGs (AREG, CXCL13, GATM, GPR171, IFI35, IFI47, IFIT3, ORM1, RETNLA, and UBD), which were enriched in immune response and cell surface receptor signalling pathway as well as interacted with immune response-related dysregulated genes during H1N1pdms. Our discoveries will provide comprehensive insights into host responding to pandemic with influenza H1N1 and find broad-spectrum effective treatment.

Emergence and genomic insights of non-pandemic O1 Vibrio cholerae in Zhejiang, China.

It is well recognized that only Vibrio cholerae O1 causes cholera pandemics. However, not all O1 strains cause pandemic-level disease. In this study, we analyzed non-pandemic O1 V. cholerae isolates from the 1960s to the 1990s from China and found that they fell into three lineages, one of which shared the most recent common ancestor with pandemic O1 strains. Each of these non-pandemic O1 lineages has unique properties that contribute to their capacity to cause cholera. The findings of this study enhanced our understanding of the emergence and evolution of both pandemic and non-pandemic O1 V. cholerae.

Incidence, genetic diversity, and antimicrobial resistance profiles of Vibrio parahaemolyticus in seafood in Bangkok and eastern Thailand.

Emergence of Vibrio parahaemolyticus pandemic strain O3:K6 was first documented in 1996. Since then it has been accounted for large outbreaks of diarrhea globally. In Thailand, prior studies on pandemic and non-pandemic V. parahaemolyticus had mostly been done in the south. The incidence and molecular characterization of pandemic and non-pandemic strains in other parts of Thailand have not been fully characterized. This study examined the incidence of V. parahaemolyticus in seafood samples purchased in Bangkok and collected in eastern Thailand and characterized V. parahaemolyticus isolates. Potential virulence genes, VPaI-7, T3SS2, and biofilm were examined. Antimicrobial resistance (AMR) profiles and AMR genes (ARGs) were determined. V. parahaemolyticus was isolated from 190 marketed and farmed seafood samples by a culture method and confirmed by polymerase chain reaction (PCR). The incidence of pandemic and non-pandemic V. parahaemolyticus and VPaI-7, T3SS2, and biofilm genes was examined by PCR. AMR profiles were verified by a broth microdilution technique. The presence of ARGs was verified by genome analysis. V. parahaemolyticus characterization was done by multilocus sequence typing (MLST). A phylogenomic tree was built from nucleotide sequences by UBCG2.0 and RAxML softwares. All 50 V. parahaemolyticus isolates including 21 pathogenic and 29 non-pathogenic strains from 190 samples had the toxRS/old sequence, indicating non-pandemic strains. All isolates had biofilm genes (VP0950, VP0952, and VP0962). None carried T3SS2 genes (VP1346 and VP1367), while VPaI-7 gene (VP1321) was seen in two isolates. Antimicrobial susceptibility profiles obtained from 36 V. parahaemolyticus isolates revealed high frequency of resistance to colistin (100%, 36/36) and ampicillin (83%, 30/36), but susceptibility to amoxicillin/clavulanic acid and piperacillin/tazobactam (100%, 36/36). Multidrug resistance (MDR) was seen in 11 isolates (31%, 11/36). Genome analysis revealed ARGs including blaCARB (100%, 36/36), tet(34) (83%, 30/36), tet(35) (42%, 15/36), qnrC (6%, 2/36), dfrA6 (3%, 1/36), and blaCTX-M-55 (3%, 1/36). Phylogenomic and MLST analyses classified 36 V. parahaemolyticus isolates into 5 clades, with 12 known and 13 novel sequence types (STs), suggesting high genetic variation among the isolates. Although none V. parahaemolyticus strains isolated from seafood samples purchased in Bangkok and collected in eastern Thailand were pandemic strains, around one third of isolates were MDR V. parahaemolyticus strains. The presence of resistance genes of the first-line antibiotics for V. parahaemolyticus infection raises a major concern for clinical treatment outcome since these resistance genes could be highly expressed under suitable circumstances.

GRP75 as a functional element of cholix transcytosis

ABSTRACT Cholix (Chx) is secreted by non-pandemic strains of Vibrio cholerae in the intestinal lumen. For this exotoxin to induce cell death in non-polarized cells in the intestinal lamina propria, it must traverse the epithelium in the fully intact form. We identified host cell elements in polarized enterocytes associated with Chx endocytosis and apical to basal (A→B) vesicular transcytosis. This pathway overcomes endogenous mechanisms of apical vesicle recycling and lysosomal targeting by interacting with several host cell proteins that include the 75 kDa glucose-regulated protein (GRP75). Apical endocytosis of Chx appears to involve the single membrane spanning protein TMEM132A, and interaction with furin before it engages GRP75 in apical vesicular structures. Sorting within these apical vesicles results in Chx being trafficked to the basal region of cells in association with the Lectin, Mannose Binding 1 protein LMAN1. In this location, Chx interacts with the basement membrane-specific heparan sulfate proteoglycan perlecan in recycling endosomes prior to its release from this basal vesicular compartment to enter the underlying lamina propria. While the furin and LMAN1 elements of this Chx transcytosis pathway undergo cellular redistribution that are reflective of the polarity shifts noted for coatamer complexes COPI and COPII, GRP75 and perlecan fail to show these dramatic rearrangements. Together, these data define essential steps in the A→B transcytosis pathway accessed by Chx to reach the intestinal lamina propria where it can engage and intoxicate certain non-polarized cells.

Type VI secretion system mutations reduced competitive fitness of classical Vibrio cholerae biotype

The gram-negative bacterium Vibrio cholerae is the causative agent of the diarrhoeal disease cholera and is responsible for seven recorded pandemics. Several factors are postulated to have led to the decline of 6th pandemic classical strains and the rise of El Tor biotype V. cholerae, establishing the current 7th pandemic. We investigated the ability of classical V. cholerae of the 2nd and 6th pandemics to engage their type six secretion system (T6SS) in microbial competition against non-pandemic and 7th pandemic strains. We report that classical V. cholerae underwent sequential mutations in T6SS genetic determinants that initially exposed 2nd pandemic strains to microbial attack by non-pandemic strains and subsequently caused 6th pandemic strains to become vulnerable to El Tor biotype V. cholerae intraspecific competition. The chronology of these T6SS-debilitating mutations agrees with the decline of 6th pandemic classical strains and the emergence of 7th pandemic El Tor V. cholerae.

Molecular analysis on Vibrio parahaemolyticus strains isolated from patients in Nanjing, Jiangsu, 2016–2019

Objective To understand the molecular characteristics of Vibrio parahaemolyticus strains in Nanjing, Jiangsu, and provide evidence for the prevention and control of human infection with V. parahaemolyticus. Methods Clinical isolates from diarrhea and food poisoning patients were collected from 2016 to 2019. Virulence genes of tlh, tdh, trh, toxRS/new and ORF8 were detected by polymerase chain reaction (PCR). Molecular types were analyzed by multilocus sequence typing (MLST). Results A total of 93 V. parahaemolyticus strains were collected. O3∶K6 was the predominant serotype (65 strains, 69.9%). Virulence genes screening revealed that all isolates carried tlh gene. 73 strains with tdh+trh-toxRS/new+ were classified as pandemic strains, and 5 non-pandemic strains with tdh-trh- were non-pathogenic strains. A total of 13 STs were detected, of which ST3 was predominant (68/93, 73.1%). Conclusion Molecular analysis on V. parahaemolyticus revealed that ST3 was predominant, and both the pandemic and non-pandemic strains were distributed in Nanjing.

Few amino acid signatures distinguish HIV-1 subtype B pandemic and non-pandemic strains.

The Human Immunodeficiency Virus Type I (HIV-1) subtype B comprises approximately 10% of all HIV infections in the world. The HIV-1 subtype B epidemic comprehends a pandemic variant (named BPANDEMIC) disseminated worldwide and non-pandemic variants (named BCAR) that are mostly restricted to the Caribbean. The goal of this work was the identification of amino acid signatures (AAs) characteristic to the BCAR and BPANDEMIC variants. To this end, we analyzed HIV-1 subtype B full-length (n = 486) and partial (n = 814) genomic sequences from the Americas classified within the BCAR and BPANDEMIC clades and reconstructed the sequences of their most recent common ancestors (MRCA). Analysis of contemporary HIV-1 sequences revealed 13 AAs between BCAR and BPANDEMIC variants (four on Gag, three on Pol, three on Rev, and one in Vif, Vpu, and Tat) of which only two (one on Gag and one on Pol) were traced to the MRCA. All AAs correspond to polymorphic sites located outside essential functional proteins domains, except the AAs in Tat. The absence of stringent AAs inherited from their ancestors between modern BCAR and BPANDEMIC variants support that ecological factors, rather than viral determinants, were the main driving force behind the successful spread of the BPANDEMIC strain.

Experimental Adaptive Evolution of Simian Immunodeficiency Virus SIVcpz to Pandemic Human Immunodeficiency Virus Type 1 by Using a Humanized Mouse Model.

Human immunodeficiency virus type 1 (HIV-1), the causative agent of AIDS, originated from simian immunodeficiency virus from chimpanzees (SIVcpz), the precursor of the human virus, approximately 100 years ago. This indicates that HIV-1 has emerged through the cross-species transmission of SIVcpz from chimpanzees to humans. However, it remains unclear how SIVcpz has evolved into pandemic HIV-1 in humans. To address this question, we inoculated three SIVcpz strains (MB897, EK505, and MT145), four pandemic HIV-1 strains (NL4-3, NLCSFV3, JRCSF, and AD8), and two nonpandemic HIV-1 strains (YBF30 and DJO0131). Humanized mice infected with SIVcpz strain MB897, a virus phylogenetically similar to pandemic HIV-1, exhibited a peak viral load comparable to that of mice infected with pandemic HIV-1, while peak viral loads of mice infected with SIVcpz strain EK505 or MT145 as well as nonpandemic HIV-1 strains were significantly lower. These results suggest that SIVcpz strain MB897 is preadapted to humans, unlike the other SIVcpz strains. Moreover, viral RNA sequencing of MB897-infected humanized mice identified a nonsynonymous mutation in env, a G413R substitution in gp120. The infectivity of the gp120 G413R mutant of MB897 was significantly higher than that of parental MB897. Furthermore, we demonstrated that the gp120 G413R mutant of MB897 augments the capacity for viral replication in both in vitro cell cultures and humanized mice. Taken together, this is the first experimental investigation to use an animal model to demonstrate a gain-of-function evolution of SIVcpz into pandemic HIV-1.IMPORTANCE From the mid-20th century, humans have been exposed to the menace of infectious viral diseases, such as severe acute respiratory syndrome coronavirus, Ebola virus, and Zika virus. These outbreaks of emerging/reemerging viruses can be triggered by cross-species viral transmission from wild animals to humans, or zoonoses. HIV-1, the causative agent of AIDS, emerged by the cross-species transmission of SIVcpz, the HIV-1 precursor in chimpanzees, around 100 years ago. However, the process by which SIVcpz evolved to become HIV-1 in humans remains unclear. Here, by using a hematopoietic stem cell-transplanted humanized-mouse model, we experimentally recapitulate the evolutionary process of SIVcpz to become HIV-1. We provide evidence suggesting that a strain of SIVcpz, MB897, preadapted to infect humans over other SIVcpz strains. We further demonstrate a gain-of-function evolution of SIVcpz in infected humanized mice. Our study reveals that pandemic HIV-1 has emerged through at least two steps: preadaptation and subsequent gain-of-function mutations.

Genetic Analysis of Vibrio parahaemolyticus O3:K6 Strains That Have Been Isolated in Mexico Since 1998.

Vibrio parahaemolyticus is an important human pathogen that has been isolated worldwide from clinical cases, most of which have been associated with seafood consumption. Environmental and clinical toxigenic strains of V. parahaemolyticus that were isolated in Mexico from 1998 to 2012, including those from the only outbreak that has been reported in this country, were characterized genetically to assess the presence of the O3:K6 pandemic clone, and their genetic relationship to strains that are related to the pandemic clonal complex (CC3). Pathogenic tdh+ and tdh+/trh+ strains were analyzed by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Also, the entire genome of a Mexican O3:K6 strain was sequenced. Most of the strains were tdh/ORF8-positive and corresponded to the O3:K6 serotype. By PFGE and MLST, there was very close genetic relationship between ORF8/O3:K6 strains, and very high genetic diversities from non-pandemic strains. The genetic relationship is very close among O3:K6 strains that were isolated in Mexico and sequences that were available for strains in the CC3, based on the PubMLST database. The whole-genome sequence of CICESE-170 strain had high similarity with that of the reference RIMD 2210633 strain, and harbored 7 pathogenicity islands, including the 4 that denote O3:K6 pandemic strains. These results indicate that pandemic strains that have been isolated in Mexico show very close genetic relationship among them and with those isolated worldwide.

Norovirus Polymerase Fidelity Contributes to Viral Transmission In Vivo.

Intrahost genetic diversity and replication error rates are intricately linked to RNA virus pathogenesis, with alterations in viral polymerase fidelity typically leading to attenuation during infections in vivo. We have previously shown that norovirus intrahost genetic diversity also influences viral pathogenesis using the murine norovirus model, as increasing viral mutation frequency using a mutagenic nucleoside resulted in clearance of a persistent infection in mice. Given the role of replication fidelity and genetic diversity in pathogenesis, we have now investigated whether polymerase fidelity can also impact virus transmission between susceptible hosts. We have identified a high-fidelity norovirus RNA-dependent RNA polymerase mutant (I391L) which displays delayed replication kinetics in vivo but not in cell culture. The I391L polymerase mutant also exhibited lower transmission rates between susceptible hosts than the wild-type virus and, most notably, another replication defective mutant that has wild-type levels of polymerase fidelity. These results provide the first experimental evidence that norovirus polymerase fidelity contributes to virus transmission between hosts and that maintaining diversity is important for the establishment of infection. This work supports the hypothesis that the reduced polymerase fidelity of the pandemic GII.4 human norovirus isolates may contribute to their global dominance. IMPORTANCE Virus replication fidelity and hence the intrahost genetic diversity of viral populations are known to be intricately linked to viral pathogenesis and tropism as well as to immune and antiviral escape during infection. In this study, we investigated whether changes in replication fidelity can impact the ability of a virus to transmit between susceptible hosts by the use of a mouse model for norovirus. We show that a variant encoding a high-fidelity polymerase is transmitted less efficiently between mice than the wild-type strain. This constitutes the first experimental demonstration that the polymerase fidelity of viruses can impact transmission of infection in their natural hosts. These results provide further insight into potential reasons for the global emergence of pandemic human noroviruses that display alterations in the replication fidelity of their polymerases compared to nonpandemic strains.

Major tdh+Vibrio parahaemolyticus serotype changes temporally in the Bay of Bengal estuary of Bangladesh

Vibrio parahaemolyticus is responsible for seafood-related gastroenteritis worldwide. In Bangladesh, diarrhea is endemic and diarrheagenic V. parahaemolyticus serotypes occur naturally in the coastal and estuarine aquatic environment. V. parahaemolyticus strains, isolated from estuarine surface water of the Bay of Bengal villages of Bangladesh during 2006–2008, were tested for the presence of virulence and pandemic-marker genes, serodiversity, and phylogenetic relatedness. PCR analysis of V. parahaemolyticus (n=175) showed 53 (30.3%) strains to possess tdh, the major virulence gene encoding thermostable direct hemolysin. Serotyping results revealed the tdh+V. parahaemolyticus strains to belong to 10 different serotypes, of which the O8:K21 (30.2%) and O3:K6 (24.5%) were predominantly non-pandemic and pandemic serotypes, respectively; while O5:K30 and O9:KUT were new. The pandemic markers, orf8 and toxRSvariant, were present only in the pandemic serotype O3:K6 (n=13) and its serovariant O4:K68 (n=2). Temporal distribution of the tdh+ serotypes revealed the O8:K21 to be predominant in 2006 and 2007, while O3:K6 was the predominant tdh+ serotype in 2008. Pulsed-field gel electrophoresis (PFGE) of SfiI-digested genomic DNA revealed high genetic diversity among the V. parahaemolyticus strains, while dendrogram constructed with the PFGE patterns formed two major clusters separating the tdh+ O3:K6 and its pandemic serovariants from the tdh+ non-pandemic (O8:K21) strains, suggesting different lineages for them. The potential health risk related to the prevalent tdh+ strains, including the observed temporal change of the predominant tdh+ serotype, from O8:K21 to the pandemic serotype O3:K6 in estuarine surface waters serving as the major source of drinking water suggests the need for routine environmental monitoring to prevent V. parahaemolyticus infection in Bangladesh.

Vibrio parahaemolyticus strengthens their virulence through modulation of cellular reactive oxygen species in vitro.

Vibrio parahaemolyticus (Vp) is one of the emergent food-borne pathogens that are commensally associated with various shellfish species throughout the world. It is strictly environmental and many strains are pathogenic to humans. The virulent strains cause distinct diseases, including wound infections, septicemia, and most commonly, acute gastroenteritis, which is acquired through the consumption of raw or undercooked seafood, especially shellfish. Vp has two type three secretion systems (T3SSs), which triggering its cytotoxicity and enterotoxicity via their effectors. To better understand the pathogenesis of Vp, we established a cell infection model in vitro using a non-phagocytic cell line. Caco-2 cells were infected with different strains of Vp (pandemic and non-pandemic strains) and several parameters of cytotoxicity were measured together with adhesion and invasion indices, which reflect the pathogen's virulence. Our results show that Vp adheres to cell monolayers and can invade non-phagocytic cells. It also survives and persists in non-phagocytic cells by modulating reactive oxygen species (ROS), allowing its replication, and resulting in complete cellular destruction. We conclude that the pathogenicity of Vp is based on its capacities for adhesion and invasion. Surprisingly's; enhanced of ROS resistance period could promote the survival of Vp inside the intestinal tract, facilitating tissue infection by repressing the host's oxidative stress response.

Spatiotemporal dynamics of dissemination of non-pandemic HIV-1 subtype B clades in the Caribbean region.

The Human immunodeficiency virus type-1 (HIV-1) epidemic in the Caribbean region is mostly driven by subtype B; but information about the pattern of viral spread in this geographic region is scarce and different studies point to quite divergent models of viral dissemination. In this study, we reconstructed the spatiotemporal and population dynamics of the HIV-1 subtype B epidemic in the Caribbean. A total of 1,806 HIV-1 subtype B pol sequences collected from 17 different Caribbean islands between 1996 and 2011 were analyzed together with sequences from the United States (n = 525) and France (n = 340) included as control. Maximum Likelihood phylogenetic analyses revealed that HIV-1 subtype B infections in the Caribbean are driven by dissemination of the pandemic clade (BPANDEMIC) responsible for most subtype B infections across the world, and older non-pandemic lineages (BCAR) characteristics of the Caribbean region. The non-pandemic BCAR strains account for >40% of HIV-1 infections in most Caribbean islands; with exception of Cuba and Puerto Rico. Bayesian phylogeographic analyses indicate that BCAR strains probably arose in the island of Hispaniola (Haiti/Dominican Republic) around the middle 1960s and were later disseminated to Trinidad and Tobago and to Jamaica between the late 1960s and the early 1970s. In the following years, the BCAR strains were also disseminated from Hispaniola and Trinidad and Tobago to other Lesser Antilles islands at multiple times. The BCAR clades circulating in Hispaniola, Jamaica and Trinidad and Tobago appear to have experienced an initial phase of exponential growth, with mean estimated growth rates of 0.35–0.45 year−1, followed by a more recent stabilization since the middle 1990s. These results demonstrate that non-pandemic subtype B lineages have been widely disseminated through the Caribbean since the late 1960s and account for an important fraction of current HIV-1 infections in the region.

Trends in the epidemiology of pandemic and non-pandemic strains of Vibrio parahaemolyticus isolated from diarrheal patients in Kolkata, India.

A total of 178 strains of V. parahaemolyticus isolated from 13,607 acute diarrheal patients admitted in the Infectious Diseases Hospital, Kolkata has been examined for serovar prevalence, antimicrobial susceptibility and genetic traits with reference to virulence, and clonal lineages. Clinical symptoms and stool characteristics of V. parahaemolyticus infected patients were analyzed for their specific traits. The frequency of pandemic strains was 68%, as confirmed by group-specific PCR (GS-PCR). However, the prevalence of non-pandemic strains was comparatively low (32%). Serovars O3:K6 (19.7%), O1:K25 (18.5%), O1:KUT (11.2%) were more commonly found and other serovars such as O3:KUT (6.7%), O4:K8 (6.7%), and O2:K3 (4.5%) were newly detected in this region. The virulence gene tdh was most frequently detected in GS-PCR positive strains. There was no association between strain features and stool characteristics or clinical outcomes with reference to serovar, pandemic/non-pandemic or virulence profiles. Ampicillin and streptomycin resistance was constant throughout the study period and the MIC of ampicillin among selected strains ranged from 24 to >256 µg/ml. Susceptibility of these strains to ampicillin increased several fold in the presence of carbonyl cyanide-m-chlorophenyldrazone. The newly reported ESBL encoding gene from VPA0477 was found in all the strains, including the susceptible ones for ampicillin. However, none of the strains exhibited the β-lactamase as a phenotypic marker. In the analysis of pulsed-field gel electrophoresis (PFGE), the pandemic strains formed two different clades, with one containing the newly emerged pandemic strains in this region.

Human immunodeficiency virus type 1 (HIV-1) subtype B epidemic in Panama is mainly driven by dissemination of country-specific clades.

The Human immunodeficiency virus type-1 (HIV-1) subtype B is the most predominant clade in Central America; but information about the evolutionary history of this virus in this geographic region is scarce. In this study, we reconstructed the spatiotemporal and population dynamics of the HIV-1 subtype B epidemic in Panama. A total of 761 HIV-1 subtype B pol sequences obtained in Panama between 2004 and 2013 were combined with subtype B pol sequences from the Americas and Europe. Maximum Likelihood phylogenetic analyses revealed that HIV-1 subtype B infections in Panama derived from the dissemination of multiple founder viruses. Most Panamanian subtype B viruses (94.5%) belong to the pandemic viral strain proposed as originated in the US, whereas others (5.5%) were intermixed among non-pandemic Caribbean strains. The bulk (76.6%) of subtype B sequences from Panama grouped within 12 country-specific clades that were not detected in other Central American countries. Bayesian coalescent-based analyses suggest that most Panamanian clades probably originated between the early 1970s and the early 1980s. The root location of major Panamanian clades was traced to the most densely populated districts of Panama province. Major Panamanian clades appear to have experienced one or two periods of exponential growth of variable duration between the 1970s and the 2000s, with median growth rates from 0.2 to 0.4 year− 1. Thus, the HIV-1 subtype B epidemic in Panama is driven by the expansion of local viral strains that were introduced from the Caribbean and other American countries at an early stage of the AIDS pandemic.

Serotype, Virulence, and Genetic Traits of Foodborne and ClinicalVibrio parahaemolyticusIsolates in Shanghai, China

Vibrio parahaemolyticus is a major foodborne pathogen in China and other countries. In this study, a total of 578 clinical V. parahaemolyticus strains and 51 foodborne strains were isolated during the period from 2009 to 2011 in the eastern coastal city of Shanghai, China. Their serotypes, virulence genes, pandemic traits, and genotyping were investigated. A total of nine O groups and 20 K types were identified by serological analysis of all isolates. Six different O groups and 14 different K types were detected among the 578 clinical strains. Eight different O groups and five K types were detected among the 51 foodborne strains. The O3:K6 serotype was the dominant serotype. A total of 200 representative clinical strains and 51 foodborne isolates were analyzed for virulence genes, pandemic traits, and genotyping. Of the clinical strains, 92.5% had the virulence genes tdh and/or trh. Four foodborne isolates had virulence genes; one trh-positive strain was O3:K6 and three tdh-positive strains were either O4:KUT or O3:KUT. Molecular typing by pulsed-field gel electrophoresis also showed divergence among the nonpandemic strains, although the pandemic strains formed a cluster. These results suggest high serodiversity and genetic diversity of V. parahaemolyticus. Pathogenic isolates were present in food, thus representing a public health risk and warranting epidemiological and ecological monitoring to ensure safety.

Molecular Epidemiology and Genetic Variation of Pathogenic Vibrio parahaemolyticus in Peru

Vibrio parahaemolyticus is a foodborne pathogen that has become a public health concern at the global scale. The epidemiological significance of V. parahaemolyticus infections in Latin America received little attention until the winter of 1997 when cases related to the pandemic clone were detected in the region, changing the epidemic dynamics of this pathogen in Peru. With the aim to assess the impact of the arrival of the pandemic clone on local populations of pathogenic V. parahaemolyticus in Peru, we investigated the population genetics and genomic variation in a complete collection of non-pandemic strains recovered from clinical sources in Peru during the pre- and post-emergence periods of the pandemic clone. A total of 56 clinical strains isolated in Peru during the period 1994 to 2007, 13 strains from Chile and 20 strains from Asia were characterized by Multilocus Sequence Typing (MLST) and checked for the presence of Variable Genomic Regions (VGRs). The emergence of O3:K6 cases in Peru implied a drastic disruption of the seasonal dynamics of infections and a shift in the serotype dominance of pathogenic V. parahaemolyticus. After the arrival of the pandemic clone, a great diversity of serovars not previously reported was detected in the country, which supports the introduction of additional populations cohabitating with the pandemic group. Moreover, the presence of genomic regions characteristic of the pandemic clone in other non-pandemic strains may represent early evidence of genetic transfer from the introduced population to the local communities. Finally, the results of this study stress the importance of population admixture, horizontal genetic transfer and homologous recombination as major events shaping the structure and diversity of pathogenic V. parahaemolyticus.

Isolation and Characterization of Pandemic and Nonpandemic Strains ofVibrio parahaemolyticusfrom an Outbreak of Diarrhea in North 24 Parganas, West Bengal, India

Strains of the enteric pathogen Vibrio parahaemolyticus harboring the thermostable hemolysin (TDH) encoding gene tdh is known to cause epidemic and pandemic diarrhea. In industrialized countries, this pathogen causes sporadic or outbreaks of diarrheal illness associated with consumption of raw or improperly cooked seafood. This report describes a foodborne outbreak of gastroenteritis caused by V. parahaemolyticus in June 2011 following consumption of food served at a funeral reception held at Habra, North 24 Parganas, West Bengal, India. About 650 people attended the function, of whom 44 had acute watery diarrhea with other clinical symptoms; 35 of them were admitted to the District Hospital for the rehydration treatment. Stool specimens collected from three hospitalized cases were positive for V. parahaemolyticus, of which two strains were identified as an O4:K8 serovar and one was identified as O3:K6 serovar. The O3:K6 strain also possessed the pandemic group-specific toxRS gene target (GS), whereas the O4:K8 strains were negative. All strains were polymerase chain reaction-positive for tdh but were polymerase chain reaction-negative for trh. All of the strains were resistant to ampicillin but were pansensitive to other antimicrobials tested. Pulsed-field gel electrophoresis (PFGE) analysis using NotI showed that the O3:K6 strain was similar to that of a recent clinical strain from Kolkata, but had diverged from other strains during previous years. In contrast, PFGE analysis showed that the O4:K8 strains were closely related but differed from the Kolkata strain.

Down-Modulation of CD8αβ Is a Fundamental Activity of Primate Lentiviral Nef Proteins

It is well established that the Nef proteins of human and simian immunodeficiency viruses (HIV and SIV) modulate major histocompatibility complex class I (MHC-I) cell surface expression to protect infected cells against lysis by cytotoxic T lymphocytes (CTLs). Recent data supported the observation that Nef also manipulates CTLs directly by down-modulating CD8αβ (J. A. Leonard, T. Filzen, C. C. Carter, M. Schaefer, and K. L. Collins, J. Virol. 85:6867-6881, 2011), but it remained unknown whether this Nef activity is conserved between different lineages of HIV and SIV. In this study, we examined a total of 42 nef alleles from 16 different primate lentiviruses representing most major lineages of primate lentiviruses, as well as nonpandemic HIV-1 strains and the direct precursors of HIV-1 (SIVcpz and SIVgor). We found that the vast majority of these nef alleles strongly down-modulate CD8β in human T cells. Primate lentiviral Nefs generally interacted specifically with the cytoplasmic tail of CD8β, and down-modulation of this receptor was dependent on the conserved dileucine-based motif and two adjacent acidic residues (DD/E) in the C-terminal flexible loop of SIV Nef proteins. Both of these motifs are known to be important for the interaction of HIV-1 Nef with AP-2, and they were also shown to be critical for down-modulation of CD4 and CD28, but not MHC-I, by SIV Nefs. Our results show that down-modulation of CD4, CD8β, and CD28 involves largely overlapping (but not identical) domains and is most likely dependent on conserved interactions of primate lentiviral Nefs with cellular adaptor proteins. Furthermore, our data demonstrate that Nef-mediated down-modulation of CD8αβ is a fundamental property of primate lentiviruses and suggest that direct manipulation of CD8+ T cells plays a relevant role in viral immune evasion.

Type III Secretion Is Essential for the Rapidly Fatal Diarrheal Disease Caused by Non-O1, Non-O139 Vibrio cholerae

ABSTRACTCholera is a severe diarrheal disease typically caused by O1 serogroup strains of Vibrio cholerae. The pathogenicity of all pandemic V. cholerae O1 strains relies on two critical virulence factors: cholera toxin, a potent enterotoxin, and toxin coregulated pilus (TCP), an intestinal colonization factor. However, certain non-O1, non-O139 V. cholerae strains, such as AM-19226, do not produce cholera toxin or TCP, yet they still cause severe diarrhea. The molecular basis for the pathogenicity of non-O1, non-O139 V. cholerae has not been extensively characterized, but many of these strains encode related type III secretion systems (TTSSs). Here, we used infant rabbits to assess the contribution of the TTSS to non-O1, non-O139 V. cholerae pathogenicity. We found that all animals infected with wild-type AM-19226 developed severe diarrhea even more rapidly than rabbits infected with V. cholerae O1. Unlike V. cholerae O1 strains, which do not damage the intestinal epithelium in rabbits or humans, AM-19226 caused marked disruptions of the epithelial surface in the rabbit small intestine. TTSS proved to be essential for AM-19226 virulence in infant rabbits; an AM-19226 derivative deficient for TTSS did not elicit diarrhea, colonize the intestine, or induce pathological changes in the intestine. Deletion of either one of the two previously identified or two newly identified AM-19226 TTSS effectors reduced but did not eliminate AM-19226 pathogenicity, suggesting that at least four effectors contribute to this strain’s virulence. In aggregate, our results suggest that the TTSS-dependent virulence in non-O1, non-O139 V. cholerae represents a new type of diarrheagenic mechanism.